PROJECT SUMMARY/ABSTRACT Metastasis, the spread of cancer cells from primary tumor to distant organs, accounts for 90% of solid tumor related mortality. The metabolic changes underlying this multistep cascade remain poorly understood. Recent work from our laboratory demonstrated that oxidative stress limits melanoma metastasis. Metastasizing cells utilize the one-carbon cycle to regenerate NADPH and combat oxidative stress. This increase in the NADPH- regenerating arm of one-carbon metabolism may limit the availability of substrates for other processes within the one-carbon cycle, including intermediates for purine biosynthesis. Consistent with this hypothesis, our preliminary data has shown that metastatic nodules have significantly higher levels of AICAR compared to subcutaneous tumors, suggesting a block in the de novo purine biosynthesis pathway. AICAR, an adenosine monophosphate analogue, is an allosteric activator of AMPK. We observe increased activation of AMPK in metastatic nodules compared to subcutaneous tumors. AMPK regulates the metabolic homeostasis by switching metabolism from anabolic to catabolic state thereby increasing cell survival in nutrient scarce conditions. I hypothesize that metastasizing cells preferentially rely on the salvage pathway and upregulate AMPK signaling, thereby switching to a catabolic metabolism to survive the hostile conditions of visceral organs. My proposal uses a clinically relevant model of melanoma metastasis in which patient-derived xenografts are transplanted into immunocompromised mice. Using this system, I will dissect the metabolic cascade and identify metabolic alterations that allow metastasizing melanoma cells to survive. In aim 1 of this proposal, I will ask whether metastasizing cells preferentially utilize the nucleotide salvage pathway during metastasis. In aim 2 of this proposal, I will investigate the role of AMPK signaling in promoting survival of metastasizing cells in circulation and upon colonization of distant organs. Successful completion of these aims will uncover the metabolic adaptations which allow metastasizing cells to survive and identify actionable targets to combat metastatic spread.